The invention relates to and enzymatic-chemical method for obtaining polyhydroxylalkanoates (PHA), especially polyhydroxybutyrate (PHB), or the copolymers thereof, from biomass. The inventive method comprises chemically treating the biomass with a reducing agent that reduces the non-PHA cell constituents of the biomass. The chemical treatment is carried out before and/or after enzymatic cell disruption. The inventive method, unlike other cell disruption techniques, makes it possible to obtain polyhydroxylalkanoates from biomass with a relatively low PHA content (for example <60%) without drastically changing or degrade the polymer properties or polymer purity.
Various methods are known for the microbial production of PHA polyesters, with the best-known representative being polyhydroxybutyrate (PBH), and/or for the production of PHA copolymers, which are disclosed in the number of documents describing the present state of the technology, since PHB and other PHA have an increasing economical importance as polymers with thermal-plastic properties that can be completely biologically decomposed. For example, EP 0 015 669 A2 describes the microbial production of PHB from methanol, while the extraction of copolymers is disclosed, for example, in EP 0 304 293 A2 and EP 0 069 497 A2. For example, glucose/propionic acid are used as a substrate mixture.
Other methods for obtaining PHB with a good yield coefficient and saccharose as a substrate are disclosed, for example, in EP 0 149 744 A1, while methods for producing PHB and copolymers thereof are disclosed in DE 196 19 084 C2.
According to the present state-of-the-art, PHA-containing biomass is processed either by extraction of a dried biomass with corresponding PHB/PHA solvents, chemically by addition of cell-destroying substances or enzymatic.
The chemical extraction is generally performed with halogenated solvents. Used are solvents that are capable of dissolving large quantities of PHA (for example chloroform) or solvents that can be used to obtain very pure PHA, since only small quantities of other cell constituents dissolve in those solvents (for example in dichloroethane). For example, EP 0 15 669 A2 discloses various methods for destroying cells and subsequently extracting PHB from destroyed cells by halogenated hydrocarbons (for example, chloroform, dichloromethane, 1,2-dichloroethane and 1,2-dichloropropane). The use of halogenated solvents is also described in other references (for example, in U.S. Pat. No. 3,275,610 chloroform; 1,2-dichloroethane in EP 0 014 490 A and EP 0 015 123 A).
In order to avoid the halogenated solvents which pose health risks, other methods using safer solvents have been developed. For example, the use of cyclic carbonic acid esters (U.S. Pat. No. 410,533) or of ethyl- and/or methyl lactate (DE 27 01 278 A1, DE 195 33 459 C1 and DE 196 23 778) are known. Also disclosed is the use of acetic acid or acetic anhydride as solvents (DE 197 12 702 A1, DD 229428 A1).
The individual process steps for PHA extraction can be described as follows:
The microbial biomass is initially separated from the culture liquid by filtration, separation or centrifugation (as long as this is not a genetically modified plant biomass), followed—depending on the efficiency—by an optional mechanical cell disruption (e.g., by using a ball mill) for increasing the extraction yields. The biomass can be dried, for example, by freeze-drying or spray-drying. PHB, PHA and/or their copolymer are extracted from the dried biomass using suitable extraction means. The purity of the extracted polymers can be increased by pre-extractions of the dried biomass using solvents that do not dissolve the polyesters.
For example, EP 0 124 309 A1 describes pre-extraction of the biomass with acetone, whereas methanol is used in EP 0 058 480 A1.
Typically, in addition to the frequently required pre-extractions, large quantities of extraction agents have to be used due to the generally relatively poor solubility of PHB and in particular of the so-called short-side-chain (ssc) PHA. Separation of the highly viscous polymer solution from the remaining cell constituents poses additional problems.
Moreover, many of the employed PHB-/PHA-solvents, in particular the halogenated solvents, are suspected to pose health risks. The use, handling and storage of large quantities of solvents usually also poses an economical and also ecological problem.
For this reason, methods have been developed which operate either without any solvents or only with small quantities of solvents. With these methods, the polymer granules are not physically dissolved, but the so-called NPCM cell constituents (non-PHA cell matter), with exception of the intracellular granules, are rendered chemically or enzymatically water-soluble as much as possible. The granules can then be separated from the aqueous solution by conventional methods.
Among one of the oldest known methods for PHB and PHA extraction is, for example, the chemical cell disruption with hypochloride. With this method, almost all cell constituents, with the exception of the polymer granules, are destroyed by oxidation. However, the method has also disadvantages. Although this method leaves the outer shape of the granules intact, the macromolecules are partially damaged. This method is restricted to a small-scale laboratory environment both as a result of the severe reduction of the molar mass of the polymers as well as the release of hazardous waste water.
Another oxidation method is described in DE 694 07 177 T2. PHA is obtained, in particular from polyhydroxybutyrate-hydroxyvalerate copolymers (PHBHV), either with hydrogen peroxide (preferably) or with peracetic acid, perborate, percarbonate and/or with chlorine or chlorine-containing oxidation agents in the presence of chelate formers. The effect of the oxidation agents, in particular of peroxides, on the PHA production is described in detail in EP 669 970 A2.
With the oxidizing methods, however, not only are undesirable cell constituents, such as nucleic acids and cell wall polymers, decomposed, but the average molar mass of the PHA polymers is also undesirably reduced. Accordingly, PHB/PHA cannot be obtained in this way without degrading the quality of the desired polymer material. This is particularly disadvantageous with biomasses having a small PHA content, since the smaller the PHA content, the more has to be oxidized and the more likely damage to the polyester occurs. In addition, radicals (e.g., on the methine carbon of a poly-□-hydroxyalkonoate) can form that cross-link the macromolecules. This partial cross-linking reduces the solubility in the PHA solvents to a point where the polymer is only able to swell. Cross-linking has an adverse effect in applications where the polymer solution needs to be post-processed. In addition, partial cross-linking reduces the biological decomposability, the biological reabsorption as well as the bio-compatibility and therefore weakens important properties of the polyhydroxyalkonoates.
Known methods that avoid these disadvantages lyse the undesirable cell constituents with the help of enzymes. For example, EP 0 145 233 A2 describes using proteolytic enzymes, in part in cooperation with a phospholipase and/or with surfactants. In a method described in DE 693 11 738 T2, proposes to combine the application of heat, possibly nuclease, proteolytic enzymes and/or phospholipases and/or lysozyme, chelate formers, surfactants and oxidizing post-treatment and/or solvent re-precipitation. KR 9502866 proposes to use only, for example, pre-extraction agents, such as hot water or acetone as well as a protease under strongly alkaline conditions.
The microorganisms employed in the aforedescribed enzymatic cell disruptions [mainly Rastonia eutrophus (formerly: Alcaligenis eutrophus), Alcaligenis latus, various pseudomonas strain and the like] are known as PHA producers with a high PHA content—approximately 60–80% in dry mass, and these methods were frequently developed more or less dependent on the employed microorganisms. It is known that the polymer granules can be easily isolated, for example using just a few steps, from microorganisms with a relatively high PHA content or specific lytic properties. It is often sufficient to use between one and two enzymes, surfactants and possibly chelate formers to extract the polymers. Sometimes, an oxidizing process is sufficient to achieve a purity of between 90 and approximately 98%, in particular when microorganisms can be used that already in the initial stage have a relatively high PHA content, for example 70–80%.
The extraction of PHA from biomass with a smaller PHA content of, for example, 30–60% requires the more complex process (i.e., more steps). The complexity increases with decreasing PHA content of the biomass. The methods known today are either not suitable at all or only in a limited fashion for extracting PHA from microorganisms with low PHA content.
It has been observed that the customary, immediate cell wall lysis in a first step (for example with lysozyme) does not necessarily offer the best possibility to wash out the NPCM fraction of the biomass. This applies particularly to PHA producers with a low PHA content. A frequently observed incomplete lysis and agglomeration of the biomass, including the PHA, can then make additional purification of the PHA granules significantly more difficult as a result of the inclusion of impurities.
The capability of the microorganisms to accumulate for high PHA content has been of primary interest in their selection for PHA extraction. The areas of application have changed from previously emphasized mass production to today's specialized products made from PHA, so that nowadays a microorganism with a relatively low polymer content may be preferred because it provides different advantages. For example, such advantages may be excellent mechanical properties of the PHA, high molar mass and the like, as well as a rapid, cost-effective culturing of the strain (e.g., no sterilization of the apparatus is required, etc.).